Hypoglycemic and growth-promoting polypeptides isolated from bacitracin

ABSTRACT

This application discloses an invention comprising a polypeptide fraction isolated from commercial bacitracin and which contains most if not all of the anti-insulinase activity of bacitracin while exhibiting few if any of the antibacterial properties. The invention herein described also involves the use of this polypeptide fraction given orally or if given parenterally either together (as composite) or in sequence as a means of treating diabetics. For such purposes, the insulin and anti-insulinase polypeptide fraction may be used as separate substances or chemically linked by a wide variety of chemical linking agents well known in the art. In addition to clinical uses, the application describes a method of using the polypeptide fraction as a growth promoter substance to be added to animal feed and also a composition useful for this purpose.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 083,557,filed 8/7/87, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to a way of enhancing the pharmacologicaleffectiveness of the hormone insulin.

There now exist a number of non-insulin containing hypoglycemic agentsand these have recently been reviewed (Asmal, et al., Drugs, 28, 62-78(1984) and Wolf, et al., Diabetologia, 22, 456-463 (1982)). TheSulfonylurea drugs stimulate insulin secretion from the pancreas andexert other as yet undetermined peripheral effects; the biguanidesinhibit gluconeogenesis, intestinal glucose absorption and mitochondrialoxidation; 2-bromopalmitate, ω-methyl 2-tetradecylglycerate, B807-27 andacyl aminocarnitines interfere with carnitine palmitoyl-transferase Ienzyme which inhibits long-chain fatty acid oxidation.

Insulin is a polypeptide hormone of molecular weight 6,000 daltons whichis secreted from the pancreas and is involved in the maintenance ofglucose homeostasis.

The diabetic patient must be treated with insulin or other hypoglycemicagents when insulin is either not produced (Type I diabetes) orinappropriately utilized (Type II diabetes) in order to deter the manyharmful effects of elevated blood glucose levels and disturbedmetabolism. It is clearly recognized that a variety of pathologicaldefects may lead to the diabetic state. A review of these and thecurrent strategies for treatment were recently reported (DiabetesDialogue, Am. J. Med., 79 (Suppl 2B), 1-44 (1985)).

Many diabetics require insulin injections one or more times a day tomaintain their blood glucose levels within normal limits after meals.

Because injected insulin is quickly degraded by tissue proteinases, itseffectiveness in lowering blood sugar is temporary. The destruction ofinsulin by the protease insulinase after injection occurs with a t_(1/2)of approximately 40 minutes and, because of this short duration ofaction, many methods have been sought to inhibit this proteolyticinactivation of insulin in order to prolong its effectiveness. It hasbeen reported in the literature that one such agent to inhibitinsulinase in vitro is the antibiotic bacitracin.

Bacitracin is a polypeptide antibiotic which has been found useful as anantimicrobial agent in humans and as a growth promoter in animals. Theantimicrobial properties to commercial bacitracin resides mainly in onetraction, bacitracin A. It works best against gram-positive bacteria bydisturbing their membrane function, cell wall synthesis and proteinmetabolism.

The most widely used food additives for growth promotion are the zincbacitracin and bacitracin methyldisalicylate forms of this antibrotic.While it is generally accepted that the addition of low levels ofantibiotics to feeds can improve animal growth and product, themechanism of action is unknown (Froyshov, Drugs and PharmaceuticalScience, Chapt. 24, 665-694 (1984)).

It has been known for many years that the commercial preparation is amixture of at least 20 different compounds with bacitracins A and Frepresenting respectively 65-70% and 15-20% of the total content (Tsuji,et al., J. Chromatog., 99, 597-608 (1974)). In addition to itsantibiotic properties, the commercial preparation of bacitracin has beenshown to be capable of inhibiting the degradation of insulin in avariety of in vitro experimental situations such as with isolated rathepatocytes and adipocytes (Duckworth, et al., Endocrinology, 108,1142-1147 (1981), Roth, et al., Biochem. Biophys. Res. Commun., 98,431-438 (1981) and Peavy, et al., Diabetes, 34, 217-221 (1985)). Thusbacitracin was shown to potentiate the action of insulin on glucoseutilization.

SUMMARY OF THE INVENTION

In accordance with the present invention there has been isolated fromcommercial USP bacitracin a fraction, comprising less than about 20%,preferably less than about 10% and most preferably less than about 5%,of the weight of the bacitracin. This fraction is apparently notantibiotically active but is disproportionately high in anti-insulinaseactivity relative to the starting bacitracin mixture.

It can be administered by itself which is alone adequate in some typesof diabetes where the body manufactures enough insulin. In mostinstances, however, this material will be administered along withinsulin, either admixed therewith or covalently linked thereto. Theinsulin can be pre-modified with other protease inhibitors prior to itsadmixture or reaction with the novel bacitracin fraction.

In accordance with yet another aspect of the invention, the novelbacitracin fraction can be used in animal feeds as a growth promoter.Bacitracin is known to enhance animal growth but is undesirable ingeneral feeds because its widespread use will result in exposure ofanimals and consumers of animals to bacteria resistant to bacitracin.Use of the non-antibiotic fraction overcomes this problem.

Apart from its growth promotion, the active fraction is itselfhypoglycaemic. Apparently it keeps insulin within the human or animalbody for a longer period of time, i.e. inhibits its decomposition.

The use of the active insulinase inhibitor fraction will overcome theproblems associated with the antibiotic resistance which sometimesdevelops in individuals exposed to food products containing traceamounts of antibiotics in the food they consume.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

To obtain the desired polypeptide fraction, bacitracin is produced inconventional manner. The principal components have been designatedbacitracin A and F, but these exhibit substantially no anti-insulinaseactivity. Accordingly, the bacitracin is subjected to fractionation invarious ways and the non-A, non-F component constitutes the desiredmaterial which can be further fractionated and/or purified.

The fractionations advantageously involve selective or preferentialadsorption and/or elution from conventional adsorbents such asSepharose, activated carbon, ion exchangers, and the like, employingselective solvents and eluants, buffers, etc. Thus which fraction willbe held more tenaciously can be adjusted by choice of conditions.

The recovered fractions are isolated and those not exhibitingsubstantial anti-insulinase activity are discarded. The desired fractionis believed to have a molecular weight of about 1400, approximately thesame as bacitracin A or F. Their anti-insulinase and antibacterialactivities can be assayed in conventional manner. For example, theinsulinase inhibitory activity was measured according to the method ofDuckworth, et al., Proc. Nat. Acad. Sci., 69, 3698-3702 (1972) and theanti-bacterial activity by the method of Patrick, et al., Antibioticsand Chemotherapy, 1, 133-137 (1951).

Administration of this purified fraction will combat insulinase in asubject's system. Thus, in the case of patients whose bodies produceenough insulin but whose problem is excessive insulinase, this materialitself may suffice. However, where insulin injections are required, thepurified fraction should be administered either prior to or along withinsulin. This can be at different times and in varying proportionsdepending on the patient's condition.

The materials are advantageously administered together and, further,advantageously covalently coupled to one another in a manner which doesnot destroy their effectiveness.

The insulin is advantageously present in about 1 to 1 and preferablyabout 0.2 to 20 times the weight of the polypeptide fraction.

When present, the covalent link can be derived from a polyvalent,preferably divalent, compound such as a dicarboxylic acid, a diamine, anaminocarboxylic acid, a dialdehyde, a diimide, a diisocyanate, etc. Thelength of the chain can vary but advantageously is from about 4 to 20atoms long. Suitable couplers include, for example, ε-aminocaproic acid,glutaraldehyde, carbodiimide, hexamethylene diisocyanate, phosgene, andthe like. A variety of available methods have been reported andcollected into several recent reviews (Kennedy et al, Clinica Chem.Acta, 70, 1-31 (1976); Method Enzymology 112, Pt. A (1985) Ch. 16, 17,19, 20, 21, 23).

The amount of couplers can be varied widely from none up to about 10% oreven 20% by weight of the combined insulin and polypeptide fraction.Such coupling can be effected in conventional manner either by mixingall three ingredients in a solvent or stepwise by first reacting thecoupler with one component and then reacting the coupled intermediatewith the other component. Then the desired material is freed ofby-products and/or impurities and is ready for use, alone or withconventional additives such as buffers, fillers, solvents, stabilizers,etc.

The novel fraction can be administered orally, parenterally or by anyother suitable means of treatment. In all cases, administration may beeither concurrent with insulin treatment, prior to or following anappropriate dose of insulin.

The exact dosage will depend on the patient's condition, body weight,etc. About 1 to 100 units and preferably about 2 to 20 units of insulinper day is suitable for an adult, although this may be exceeded ordiminished in special cases, as called for by the physician.

When the polypeptide fraction is used as an animal feed supplement, itcan be present in from about 1 to 100 ppm and preferably about 3 to 50ppm by weight but can be further diluted by the animal's feed. Theamount administered depends upon the animal and its stage ofdevelopment.

The invention will be further described in the following exampleswherein all parts are by weight unless otherwise expressed.

EXAMPLE 1

A 350 ml bed volume of CM-Sepharose in a packed column (2.8×56 cm) wasequilibrated with 0.05M ammonium acetate, pH 4.5 One gram of commercialbacitracin dissolved in 50 ml of buffer was applied to the packedcolumn. The material was eluted with 2:1 of equilibrating bufferfollowed by 350 ml of 1M ammonium acetate pH 4.5 buffer. Five mlfractions were collected and absorbance measurements at 252, 280 and 290nm were made. In addition, insulinase inhibitory activity measurementswere made on each peak. Bacitracins F and A, exhibiting negligibleinsulinase inhibitory activity, were eluted as distinct peaks with thefirst buffer. The peaks were collected, lyophilized, weighed anddissolved in water at 5 mg/ml concentration for specific activitymeasurements. The material collected with the second buffer contained5-10 % of the total weight of the applied material and 90 % of the totalinsulinase inhibitory capacity. The specific activity of this materialwas 10-12 times that of the starting commercial material. HPLC and gelfiltration studies revealed that this fraction contains severalcomponents of varying insulinase inhibitory capacity.

EXAMPLE 2

10 μmol (mw=1400 approximately)) of the active fraction of Example 1 aredissolved in 1 ml dry dimethylformamide (DMF). To this 20 μmol1-ethyl-3,3 dimethylaminopropylcarbodiimide (CDI) and 2 μmol ofN-hydroxysuccinimide are added. The mass is permitted to react for 2hours at room temperature, and then the product is coupled with insulinby adding, in a dropwise manner with stirring, 1.5 μmoles of insulindissolved in 30 ml of 0.05 M sodium acetate solution. After 2 hours atroom temperature the product is supplied to a 50 cm chromatographiccolumn 3.5 cm in diameter filled with Sephadex LH20 support material.Elution can be effected with 0.9% NaCl solution, collecting fractions.Those fractions which show antiinsulinase activity can be usedclinically.

EXAMPLE 3

a) 10 μmol of the active fraction of Example 1 are treated with 150nmoles of N-hydroxysuccinimide and 500 μmols of carbodiimide and 70μmoles of diaminobutane. The reaction is allowed to proceed at roomtemperature for 4 hours and the mixture is separated by Sephadexchromatography as in Example 2 and the various fractions are tested foramino functional groups with picrylsulfonic acid. The firstamino-containing fraction is the diaminobutane derivative of bacitracin.This fraction is lyophylized.

b) 10 μmoles of insulin are dissolved in 0.01 M HCl to a concentrationof 0.6 mg/ml, carbodiimide is introduced to a concentration of 0.1 M andthe pH is adjusted to 7.0 by dilute NaOH. The product of (a) isdissolved in 2 cc of water and added dropwise to the insulin solutionwhere it is kept at pH 7 for 1 hour.

The products of Examples 2 and 3 can be isolated by Sephadexchromatography and the active fraction lyophilized prior toadministration of an appropriate amount.

It will be understood that the specification and examples areillustrative but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

What is claimed is:
 1. A polypeptide fraction isolated from bacitracinand, on a weight basis, exhibiting at least 5 times the anti-insulinaseactivity of the bacitracin, the fraction having a molecular weight ofabout 1400, being obtainable by adsorption from bacitracin onCM-Sepharose and subsequent elution and being substantially free of Aand F acitracin components.
 2. A composition comprising insulin and apolypepetide fraction isolated from bacitracin and, on a weight basis,exhibiting at least 5 times the anti-insulinase activity of thebacitracin, the fraction having a molecular weight of about 1400, beingobtainable by adsorption from bacitracin on CM-Sepharose and subsequentelution and being substantially free of A and F bacitracin components.3. A composition according to claim 2, wherein the insulin is present inabout 0.2 to 20 times the weight of the polypeptide fraction.
 4. Acomposition according to claim 2, wherein the insulin is covalentlylinked to the polypeptide fraction.
 5. A composition comprising acomposition according to claim 2 and a pharmaceutically acceptablediluent.
 6. A composition according to claim 4, wherein the covalentlinkage includes at least one member selected from the group consistingof a dicarboxylic acid, a diamine, an aminocarboxylic acid, adialdehyde, a diimide, a diisocyanate and a carboxylic acid dihalide. 7.A composition according to claim 4, wherein the covalent linkageincludes at least one member selected from the group consisting ofε-aminocaproic acid, glutaraldehyde, carbodiimide, hexamethylenediisocyanate and phosgene.
 8. In the administration of insulin to apatient in need thereof, the improvement which comprises administeringthe insulin along with a polypeptide fraction according to claim
 1. 9.The method according to claim 8, wherein the insulin is covalentlylinked to the polypeptide fraction.
 10. A method of treatinghyperglycaemia in a patient in need thereof, which comprisesadministering to such patient a hypoglycaemic effective amount of apolypeptide fraction according to claim 1.